Countering Oxidative Stress In The Retina With The Ubiquitin System
The aim of this application is to test the hypothesis that the ubiquitin system can be manipulated to augment the inherent anti-oxidant system present in retinal pigment epithelial (RPE) cells and photoreceptors. The ubiquitin system is one of the methods that cells use to destroy old or unnecessary proteins, and can be used to help regulate the activities of cellular protein. This hypothesis will be tested in mice using a virus to over-express a ubiquitin conjugating enzyme (UbcM2) coupled to a well-established, light-stress model of retinal degeneration.
Cells in the eye that are essential for vision can be damaged or killed by different types of stress, including bright light. The loss of these cells causes a decrease in visual acuity and can ultimately cause loss of vision. These same cells have a defense system to protect themselves against stress. The main component of this defense system is a protein called Nrf2. The work of this proposal is intended to increase the function of Nrf2 and thereby help protect eyes from stress-induced damage. This will be accomplished using a protein called UbcM2. UbcM2 increases the amount of Nrf2 in cells. The specific aim of this study is to test the ability of extra UbcM2 to protect mice eyes from stress-induced damage. The idea being studied here is that by introducing extra UbcM2 into mouse eyes, Nrf2 levels will be increased. This increase in Nrf2 will defend the cells of the eye against stress-induced damage. This approach is novel and is anticipated to provide new strategies for developing drugs that can prevent the onset of age-related macular degeneration.
The goal of this BrightFocus-funded project is to explore a novel approach for countering oxidative stress in retinal pigment epithelial (RPE) cells and in photoreceptor visual cells. RPE cells nourish the photoreceptor cells in the retina. In simplest terms, oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) and the body's ability to eliminate or neutralize ROS. Excessive ROS irreversibly damages cells. RPE cells and photoreceptors are highly susceptible to damage by ROS and mounting clinical and animal studies strongly implicate chronic oxidative stress as a primary contributor to the cause of age-related macular degeneration (AMD).
We have developed a protein-based therapeutic treatment that increases the ability of cells to eliminate ROS and thereby reduces oxidative stress. We are targeting this protein directly to the RPE cells and photoreceptors of mice by sub-retinally injecting (delivering through a needle to a point just under the retina) adeno-associated viruses (AAVs) that express the therapeutic protein. AAVs have been used safely and successfully in eye gene therapy clinical trials. We have made significant progress during the initial phase of this project in 2009. In collaboration with Dr. William Hauswirth (at University of Florida), we have generated highly purified and concentrated AAVs that express our therapeutic protein. Further, we have sub-retinally injected the viruses into mouse eyes to determine how much time it takes for the therapeutic protein to be expressed and to establish a safety profile for expression of the protein in the retina.
Plafker KS, Farjo KM, Wiechmann AF, Plafker SM. The human ubiquitinconjugating enzyme, UBE2E3, is required for proliferation of retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 49:5611-5618, 2008.
Plafker KS, Singer JD, Plafker SM. The ubiquitin conjugating enzyme, UbcM2, engages in novel interactions with components of cullin-3 based E3 ligases. Biochemistry 48:3527-3537, 2009.
Plafker KS, Nguyen L, Barneche M, Mirza S, Crawford D, Plafker SM. The ubiquitin-conjugating enzyme UbcM2 can regulate the stability and activity of the antioxidant transcription factor Nrf2. J Biol Chem 285:23064-23074, 2010.
Plafker SM. Oxidative stress and the ubiquitin proteolytic system in age-related macular degeneration. Adv Exp Med Biol 664:447-456, 2010.
Mirza S, Plafker KS, Aston C, Plafker SM. Expression and distribution of the class III ubiquitin-conjugating enzymes in the retina. Mol Vis 16:2425- 2437, 2010.
First published on: April 15, 2009
Last modified on: August 19, 2020